The invention is directed to the detection and measurement of fluorescence of a thin layer of several 100-nm thickness of dye-labeled molecules on a glass substrate. With reference to FIG. 1, the thin layer is shown as element 1a and the glass substrate as element 2.
The primary object of the invention is to detect the fluorescence efficiency quantitatively depending on the lateral location in the layer (1a).
As shown in FIG. 1, in order for the thin, optically active layer (1a) to be subject to chemical process prior to or during optical observation, it lies on the inside of a channel (3) which is filled with liquid, for example, an aqueous solution. A process of this kind is, for example, staining the layer (1a) with a dye solution.
The channel (3) is formed by the substrate and a channel back wall (4) which is shown in section. The channel back wall (4) can be made of different materials. Plastic which is excited to fluorescence during illumination is often used. In this case, the emission of the channel back wall interferes with optical observation.
The back reflection and backscattering of excitation light in the objective (5) is also troublesome. Back reflection and backscattering take place not only at the channel back wall but also on the upper side and underside of the substrate.
In the instrument described herein in accordance with the invention, observation and excitation are carried out with an objective (5) through the glass substrate (2). The optical design of the instrument is carried out in such a way that observation is not falsified by interfering light. In particular, the optical construction of the instrument prevents the fluorescent light of the channel back wall (4) or scattered light and back-reflected excitation light from impinging on the detector surface (11a) of the instrument.